Modulation of tumor immunity

ABSTRACT

Methods of treating proliferative disorders are described. In particular, combination treatment with a GITR agonist and a PD-1 antagonist are provided.

FIELD OF THE INVENTION

The present invention relates to modulation of tumor immunity in thetreatment of advanced tumors. In particular, the present inventionprovides antagonists of PD-1 in combination with agonists of GITR toenhance anti-tumor responses to advanced tumors.

BACKGROUND OF THE INVENTION

The tumor microenvironment is an important aspect of cancer biology thatcontributes to tumor initiation, tumor progression and responses totherapy. Cells and molecules of the immune system are a fundamentalcomponent of the tumor microenvironment. Importantly, therapeuticstrategies can harness the immune system to specifically target tumorcells and this is particularly appealing owing to the possibility ofinducing tumor-specific immunological memory, which might causelong-lasting regression and prevent relapse in cancer patients.

The composition and characteristics of the tumor microenvironment varywidely and are important in determining the anti-tumor immune response.For example, certain cells of the immune system, including naturalkiller cells, dendritic cells (DCs) and effector T cells, are capable ofdriving potent anti-tumor responses. However, tumor cells often inducean immunosuppressive microenvironment, which favors the development ofimmunosuppressive populations of immune cells, such as myeloid-derivedsuppressor cells and regulatory T cells. Understanding the complexity ofimmunomodulation by tumors is important for the development ofimmunotherapy. Various strategies are being developed to enhanceanti-tumor immune responses, including DC-based vaccines and antagonistsof inhibitory signaling pathways to overcome ‘immune checkpoints’.

Glucocorticoid-induced TNFR-related protein (GITR), a member of the TNFRsuperfamily, is expressed in many components of the innate and adaptiveimmune system (see, e.g., Hanabuchi, et al. (2006) Blood 107:3617-3623;and Nocentini and Riccardi (2005) Eur. J. Immunol. 2005. 35:1016-1022).Its membrane expression is increased following T cell activation(Hanabuchi, supra; and Nocentini and Riccardi, supra); its triggeringco-activates effector T lymphocytes and modulates regulatory T cell(Treg) activity (see, e.g., McHugh, et al. (2002) Immunity 2002.16:311-323; Shimizu, et al. (2002) Nat. Immunol. 3:135-142; Ronchetti,et al. (2004) Eur. J. Immunol. 34:613-622; and Tone, et al. (2003) Proc.Natl. Acad. Sci. USA 100:15059-15064.

GITR is activated by GITR ligand (GITRL), which is mainly expressed onAPC and has been suggested to deliver signals by its cytoplasmic domain,although further studies are necessary to define the biochemicalsignaling (Nocentini, supra; Ronchetti, supra; Suvas, et al. (2005) J.Virol. 79:11935-11942; and Shin, et al. (2002) Cytokine 19:187-192).

GITR activation increases resistance to tumors and viral infections, isinvolved in autoimmune/inflammatory processes and regulates leukocyteextravasation (Nocentini supra; Cuzzocrea, et al. (2004) J. Leukoc.Biol. 76:933-940; Shevach, et al. (2006) Nat. Rev. Immunol. 6:613-618;Cuzzocrea, et al. (2006) J. Immunol. 177:631-641; and Cuzzocrea, et al.(2007) FASEB J. 21:117-129). In tumor mouse models, agonist GITRantibody, DTA-1, was combined with an antagonist CTLA-4 antibody, andshowed synergistic results in complete tumor regression of advancedstage tumors in some test group mice (Ko, et al. (2005) J. Exp. Med.7:885-891).

Programmed death receptor 1 (PD-1) is an immunoinhibitory receptor thatis primarily expressed on activated T and B cells. Interaction with itsligands has been shown to attenuate T-cell responses both in vitro andin vivo. Blockade of the interaction between PD-1 and one of itsligands, PD-L1, has been shown to enhance tumor-specific CD8+ T-cellimmunity and may therefore be helpful in clearance of tumor cells by theimmune system.

PD-1 (encoded by the gene Pdcd1) is an Immunoglobulin superfamily memberrelated to CD28, and CTLA-4. PD-1 has been shown to negatively regulateantigen receptor signaling upon engagement of its ligands (PD-L1 and/orPD-L2) The structure of murine PD-1 has been solved as well as theco-crystal structure of mouse PD-1 with human PD-L1 (Zhang, X., et al.,(2004) Immunity 20: 337-347; Lin, et al., (2008) Proc. Natl. Acad. Sci.USA 105: 3011-6). PD-1 and like family members are type I transmembraneglycoproteins containing an Ig Variable-type (V-type) domain responsiblefor ligand binding and a cytoplasmic tail that is responsible for thebinding of signaling molecules. The cytoplasmic tail of PD-1 containstwo tyrosine-based signaling motifs, an ITIM (immunoreceptortyrosine-based inhibition motif) and an ITSM (immunoreceptortyrosine-based switch motif).

In humans, expression of PD-1 (on tumor infiltrating lymphocytes) and/orPD-L1 (on tumor cells) has been found in a number of primary tumorbiopsies assessed by immunohistochemistry. Such tissues include cancersof the lung, liver, ovary, cervix, skin, colon, glioma, bladder, breast,kidney, esophagus, stomach, oral squamous cell, urothelial cell, andpancreas as well as tumors of the head and neck (Brown, J. A., et al.,(2003) J. Immunol. 170: 1257-1266; Dong H., et al., (2002) Nat. Med. 8:793-800; Wintterle, et al., (2003) Cancer Res. 63: 7462-7467; Strome, S.E., et al., (2003) Cancer Res. 63: 6501-6505; Thompson, R. H., et al.,(2006) Cancer Res. 66: 3381-5; Thompson, et al., (2007) Clin. CancerRes. 13: 1757-61; Nomi, T., et al., (2007) Clin. Cancer Res. 13:2151-7). More strikingly, PD-ligand expression on tumor cells has beencorrelated to poor prognosis of cancer patients across multiple tumortypes (reviewed in Okazaki and Honjo, (2007) Int. Immunol. 19: 813-824).

To date, numerous studies have shown that interaction of PD-1 with itsligands (PD-L1 and PD-L2) leads to the inhibition of lymphocyteproliferation in vitro and in vivo. Blockade of the PD-1/PD-L1interaction could lead to enhanced tumor-specific T-cell immunity andtherefore be helpful in clearance of tumor cells by the immune system.To address this issue, a number of studies were performed. In a murinemodel of aggressive pancreatic cancer (Nomi, T., et al. (2007) Clin.Cancer Res. 13: 2151-2157), the therapeutic efficacy of PD-1/PD-L1blockade was demonstrated. Administration of either PD-1 or PD-L1directed antibody significantly inhibited tumor growth. Antibodyblockade effectively promoted tumor reactive CD8+ T cell infiltrationinto the tumor resulting in the up-regulation of anti-tumor effectorsincluding IFN gamma, granzyme B and perforin. Additionally, the authorsshowed that PD-1 blockade can be effectively combined with chemotherapyto yield a synergistic effect. In another study, using a model ofsquamous cell carcinoma in mice, antibody blockade of PD-1 or PD-L1significantly inhibited tumor growth (Tsushima, F., et al., (2006) OralOncol. 42: 268-274).

The need exists for improved methods and compositions for the treatmentof immune and proliferative disorders, e.g., tumors and cancers, by useof agents that modulate tumor immunity. The present invention fills thisneed by providing antagonists of PD-1 in combination with agonists ofGITR to treat advanced stage tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1K shows the effect of anti-GITR antibodies dosed alone or incombination with anti-PD-1 antibodies on the anti-tumor response of miceimplanted with MC38 cell line (n=10/group). Treatment was commenced whentumors reached 240-360 mm³.

FIGS. 2A-2F show the anti-tumor efficacy of a single dose of anti-GITRantibodies followed by a single dose of anti-PD-1 antibodies one weeklater (FIG. 2B), or in the opposite sequence (FIG. 2C). This wascompared to either antibody alone (FIGS. 2E-2F; n=10/group)

FIGS. 3A-3D show the anti-tumor efficacy of monotherapy of anti-GITR oranti-PD-1 antibodies alone (FIGS. 3C-3D), compared to co-administrationof both antibodies (FIG. 3A) in the CT26 tumor model (n=10/group).

FIGS. 4A-4D show the effect of anti-GITR and anti PD-1 antibodies dosedalone or with concurrent administration of both antibodies on theanti-tumor response of mice implanted with the MB49 cell line(n=10/group). Treatment was commenced when tumors reached 85-122 mm³.

FIGS. 5A-5B show the dose dependent effect of the combination ofanti-GITR (MK-4166) and anti-PD-1 (MK-3475) on Tregs (FIG. 5A) andTreg:CD8 cell ratio (FIG. 5B) in a mixed lymphocyte reaction (MLR).

FIG. 6 shows that incubation with a combination of MK-4166 and MK-3475results in reduced suppressive activity of Tregs in an MLR.

SUMMARY OF THE INVENTION

The present invention meets these needs in the art and more by providinga method of treating a tumor in a patient comprising administering tothe patient a PD-1 antagonist and a GITR agonist, wherein the PD-1antagonist and GITR agonist are administered simultaneously orsequentially. In certain embodiments, the PD-1 antagonist is an antibodyor antigen binding fragment thereof, that binds PD-1 or PD-L1; and theGITR agonist is an antibody or antigen binding fragment thereof thatbinds GITR. The GITR agonist and PD-1 or PD-L1 antagonist binds to thehuman proteins. The antibody or binding fragment thereof is humanized orfully human.

In further embodiments, the PD-1 antagonist is selected from the groupconsisting of BMS-936558, MK-3475, and MPDL3280A; and GITR agonist isselected from the group consisting of an antibody having at least oneCDR of SEQ ID NOs: 1-66; TRX518; and TRX385. The GITR agonist can be anantibody having: a heavy chain CDR1 of SEQ ID NOs: 1-11, CDR2 of SEQ IDNOs: 12-22, and CDR3 of SEQ ID NOs: 23-33; and/or a light chain CDR1 ofSEQ ID NOs: 34-44, CDR2 of SEQ ID NOs: 45-55, and CDR3 of SEQ ID NOs:56-66. In yet a further embodiment, the GITR agonist is an antibodyhaving: a variable heavy chain of SEQ ID NOs: 67, 69, 71, 73, 75, 77,79, 81, 83, 85, and 87; and/or a variable light chain of SEQ ID NO: 68,70, 72, 74, 76, 78, 80, 82, 84, 86, and 88.

The present invention also contemplates that the PD-1 antagonist andGITR agonist are administered concurrently at least one time. In certainembodiments, the PD-1 antagonist and GITR agonist are administeredconcurrently at least 2 times. In certain embodiments, the tumor is anadvanced stage tumor and can be selected from the group consistingsquamous cell cancer, small-cell lung cancer, non-small cell lungcancer, gastrointestinal cancer, pancreatic cancer, glioblastoma,glioma, cervical cancer, ovarian cancer, liver cancer such as hepaticcarcinoma and hepatoma, bladder cancer, breast cancer, colon cancer,colorectal cancer, endometrial carcinoma, myeloma (such as multiplemyeloma), salivary gland carcinoma, kidney cancer such as renal cellcarcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostatecancer, vulval cancer, thyroid cancer, testicular cancer, and esophagealcancer.

The present invention provides method of treating a tumor, byadministering to a patient a bispecific antibody comprising a first armthat binds to PD-1 or PD-L1 and antagonizes PD-1 activity, and a secondarm that binds to GITR and agonizes GITR activity. In certainembodiments, the first arm is selected from the group consisting of anantigen binding fragment from BMS-936558, MK-3475, and MPDL3280A; andthe second arm is selected from the group consisting of an antigenbinding fragment from an antibody having at least one CDR of SEQ ID NO:1-66; TRX518; and TRX385. In yet a further embodiment, the second armhas a heavy chain CDR1 of SEQ ID NOs: 1-11, CDR2 of SEQ ID NOs: 12-22,and CDR3 of SEQ ID NOs: 23-33; and/or a light chain CDR1 of SEQ ID NOs:34-44, CDR2 of SEQ ID NOs: 45-55, and CDR3 of SEQ ID NOs: 56-66. Incertain embodiments, the second arm has a variable heavy chain of SEQ IDNOs: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, and 87; and/or a variablelight chain of SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, and88.

The present invention provides a method of treating a tumor wherein thetumor is an advanced stage tumor. In certain embodiments, the advancedstage tumor is selected from the group consisting of squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma,cervical cancer, ovarian cancer, liver cancer such as hepatic carcinomaand hepatoma, bladder cancer, breast cancer, colon cancer, colorectalcancer, endometrial carcinoma, myeloma (such as multiple myeloma),salivary gland carcinoma, kidney cancer such as renal cell carcinoma andWilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulvalcancer, thyroid cancer, testicular cancer, and esophageal cancer.

The present invention provides a pharmaceutical composition comprising aPD-1 antagonist and a GITR agonist. Also provided is the use of a PD-1antagonist in combination with a GITR agonist to treat an advanced stagetumor.

DETAILED DESCRIPTION

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise. Table 15 belowprovides a listing of sequence identifiers used in this application. Allreferences cited herein are incorporated by reference to the same extentas if each individual publication, database entry (e.g. GenBanksequences or GeneID entries), patent application, or patent, wasspecifically and individually indicated to be incorporated by reference.Citation of the references herein is not intended as an admission thatany of the foregoing is pertinent prior art, nor does it constitute anyadmission as to the contents or date of these publications or documents.

Definitions

The term “glucocorticoid-induced TNF receptor” (abbreviated herein as“GITR”), also known as TNF receptor superfamily 18 (TNFRSF18), TEASR,and 312C2, as used herein, refers to a member of the tumor necrosisfactor/nerve growth factor receptor family. GITR is a 241 amino acidtype I transmembrane protein characterized by three cysteinepseudo-repeats in the extracellular domain and specifically protectsT-cell receptor-induced apoptosis, although it does not protect cellsfrom other apoptotic signals, including Fas triggering, dexamethasonetreatment, or UV irradiation (Nocentini, G., et al. (1997) Proc. Natl.Acad. Sci. USA 94:6216-622). The nucleic acid and amino acid sequencesof human GITR (hGITR), of which there are three splice variants, areknown and can be found in, for example GenBank Accession Nos.gi:40354198, gi:23238190, gi:23238193, and gi:23238196.

“GITR agonist” means any chemical compound or biological molecule thatstimulates an immune reaction through activation of GITR signaling.Sequences of agonist anti-GITR antibodies are provided in WO 2011/028683and WO 2006/105021, as well as TRX-385 and TRX-518. Also contemplatedare soluble GITR-L proteins, a GITR binding partner.

“PD-1 antagonist” means any chemical compound or biological moleculethat blocks binding of PD-L1 expressed on a cancer cell to PD-1expressed on an immune cell (T cell, B cell or NKT cell) and preferablyalso blocks binding of PD-L2 expressed on a cancer cell to theimmune-cell expressed PD-1. Alternative names or synonyms for PD-1 andits ligands include: Programmed death receptor 1; PDCD1, PD1, CD279 andSLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1; andProgrammed death receptor Ligand 1, PDCD1L2, PDL2, B7-DC, Btdc and CD273for PD-L2. In any of the treatment method, medicaments and uses of thepresent invention in which a human individual is being treated, the PD-1antagonist blocks binding of human PD-L1 to human PD-1, and preferablyblocks binding of both human PD-L1 and PD-L2 to human PD-1. Human PD-1amino acid sequences can be found in NCBI Locus No.: NP_005009. HumanPD-L1 and PD-L2 amino acid sequences can be found in NCBI Locus No.:NP_054862 and NP_079515, respectively.

PD-1 antagonists useful in the any of the treatment method, medicamentsand uses of the present invention include a monoclonal antibody (mAb),or antigen binding fragment thereof, which specifically binds to PD-1 orPD-L1, and preferably specifically binds to human PD-1 or human PD-L1.The mAb may be a human antibody, a humanized antibody or a chimericantibody, and may include a human constant region. In some embodimentsthe human constant region is selected from the group consisting of IgG1,IgG2, IgG3 and IgG4 constant regions, and in preferred embodiments, thehuman constant region is an IgG1 or IgG4 constant region. In someembodiments, the antigen binding fragment is selected from the groupconsisting of Fab, Fab′-SH, F(ab′)₂, scFv and Fv fragments.

Examples of mAbs that bind to human PD-1, and are useful in thetreatment method, medicaments and uses of the present invention, aredescribed in U.S. Pat. No. 7,521,051, U.S. Pat. No. 8,008,449, and U.S.Pat. No. 8,354,509. Specific anti-human PD-1 mAbs useful as the PD-1antagonist in the treatment method, medicaments and uses of the presentinvention include: MK-3475, a humanized IgG4 mAb with the structuredescribed in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013)and which comprises the heavy and light chain amino acid sequences shownin FIG. 6, nivolumab (BMS-936558), a human IgG4 mAb with the structuredescribed in WHO Drug Information, Vol. 27, No. 1, pages 68-69 (2013)and which comprises the heavy and light chain amino acid sequences shownin FIG. 7; pidilizumab (CT-011, also known as hBAT or hBAT-1); and thehumanized antibodies h409A11, h409A16 and h409A17, which are describedin WO2008/156712.

Examples of mAbs that bind to human PD-L1, and are useful in thetreatment method, medicaments and uses of the present invention, aredescribed in WO2013/019906, WO2010/077634 A1 and U.S. Pat. No.8,383,796. Specific anti-human PD-L1 mAbs useful as the PD-1 antagonistin the treatment method, medicaments and uses of the present inventioninclude MPDL3280A, BMS-936559, MEDI4736, MSB0010718C and an antibodywhich comprises the heavy chain and light chain variable regions of SEQID NO:24 and SEQ ID NO:21, respectively, of WO2013/019906.

The term “administering” as used herein refers to the physicalintroduction of a composition comprising a GITR agonist and at least oneadditional cancer therapeutic agent, e.g., a PD-1 antagonist to apatient with cancer. Any and all methods of introduction arecontemplated according to the invention; the method is not dependent onany particular means of introduction. Means of introduction arewell-known to those skilled in the art, examples of which are providedherein.

The term “co-administering” as used herein means a process whereby thecombination of a GITR agonist and at least one additional cancertherapeutic agent, e.g., a PD-1 antagonist, is administered to the samepatient. The GITR agonist and PD-1 antagonist may be administeredconcurrently or sequentially. If administration takes placesequentially, the GITR agonist and/or PD-1 antagonist may beadministered before or after a given additional cancer therapeutic agentor treatment. The GITR agonist and PD-1 antagonist treatment need not beadministered by means of the same vehicle. The GITR agonist and PD-1antagonist may be administered one or more times and the number ofadministrations of each component of the combination may be the same ordifferent. In addition, GITR agonist and PD-1 antagonist need not beadministered at the same site.

The term “therapeutically effective amount” or “therapeuticallyeffective combination” as used herein refers to an amount or dose of aGITR agonist, together with the amount or dose of an additional agent ortreatment, e.g., a PD-1 antagonist that is sufficient to modulate, e.g.,stimulate, the systemic immune response of an individual. The amount ofeach molecule in a given therapeutically effective combination may bedifferent for different individuals and different tumor types, and willbe dependent upon the one or more additional agents or treatmentsincluded in the combination. The “therapeutically effective amount” isdetermined using procedures routinely employed by those of skill in theart such that an “improved therapeutic outcome” results.

As used herein, the terms “improved therapeutic outcome” and “enhancedtherapeutic efficacy,” relative to cancer refers to a slowing ordiminution of the growth of cancer cells or a solid tumor, or areduction in the total number of cancer cells or total tumor burden. An“improved therapeutic outcome” or “enhanced therapeutic efficacy”therefore means there is an improvement in the condition of the patientaccording to any clinically acceptable criteria, including, for example,decreased tumor size, an increase in time to tumor progression,increased progression-free survival, increased overall survival time, anincrease in life expectancy, or an improvement in quality of life. Inparticular, “improved” or “enhanced” refers to an improvement orenhancement of 1%, 5%, 10%, 25% 50%, 75%, 100%, or greater than 100% ofany clinically acceptable indicator of therapeutic outcome or efficacy.

As used herein, the term “antibody” refers to any form of antibody thatexhibits the desired biological activity. Thus, it is used in thebroadest sense and specifically covers monoclonal antibodies (includingfull length monoclonal antibodies), polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), chimeric antibodies, humanizedantibodies, fully human antibodies, etc. so long as they exhibit thedesired biological activity.

As used herein, the terms “GITR, PD-1, or PD-L1 binding fragment,”“binding fragment thereof” or “antigen binding fragment thereof”encompass a fragment or a derivative of an antibody that stillsubstantially retains its biological activity of inducing GITR signalingreferred to herein as “GITR inducing activity.” Alternatively, PD-1 orPD-L1 binding fragment encompasses a fragment or derivative of antibodythat inhibits PD-1 activity, e.g., binding to PD-L1 or PD-L2. The term“antibody fragment” or GITR, PD-1, or PD-L1 binding fragment refers to aportion of a full length antibody, generally the antigen binding orvariable region thereof. Examples of antibody fragments include Fab,Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules, e.g., sc-Fv; and multispecificantibodies formed from antibody fragments. Typically, a binding fragmentor derivative retains at least 10% of its GITR agonist activity.Preferably, a binding fragment or derivative retains at least 25%, 50%,60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its GITR agonist orPD-1 antagonist activity, although any binding fragment with sufficientaffinity to exert the desired biological effect will be useful. It isalso intended that a GITR, PD-1, or PD-L1 binding fragment can includevariants having conservative amino acid substitutions that do notsubstantially alter its biologic activity.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. In contrast, conventional(polyclonal) antibody preparations typically include a multitude ofantibodies directed against (or specific for) different epitopes. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler, et al. (1975) Nature 256: 495, or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson, et al. (1991)Nature 352: 624-628 and Marks, et al. (1991)J. Mol. Biol. 222: 581-597,for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity. U.S. Pat. No. 4,816,567;Morrison, et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855.

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody maytarget the same or different antigens.

A “bivalent antibody” comprises two antigen binding sites. In someinstances, the two binding sites have the same antigen specificities.However, bivalent antibodies may be bispecific (see below).

As used herein, the term “single-chain Fv” or “scFv” antibody refers toantibody fragments comprising the V_(H) and V_(L) domains of antibody,wherein these domains are present in a single polypeptide chain.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains which enables the sFv to form thedesired structure for antigen binding. For a review of sFv, seePluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.

The monoclonal antibodies herein also include camelized single domainantibodies. A “domain antibody fragment” is an immunologicallyfunctional immunoglobulin fragment containing only the variable regionof a heavy chain or the variable region of a light chain. In someinstances, two or more V_(H) regions are covalently joined with apeptide linker to create a multivalent domain antibody fragment. The twoV_(H) regions of a bivalent domain antibody fragment may target the sameor different antigens. See, e.g., Muyldermans, et al. (2001) TrendsBiochem. Sci. 26:230; Reichmann, et al. (1999) J. Immunol. Methods231:25; WO 94/04678; WO 94/25591; U.S. Pat. No. 6,005,079). In oneembodiment, the present invention provides single domain antibodiescomprising two V_(H) domains with modifications such that single domainantibodies are formed.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L) or V_(L)-V_(H)). Byusing a linker that is too short to allow pairing between the twodomains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. Diabodies are described more fully in, e.g., EP 404,097; WO93/11161; and Holliger, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For a review of engineered antibody variants generally seeHolliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies contain minimalsequence derived from non-human immunoglobulin. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable loops correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence. The humanized antibody optionallyalso will comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. The prefix “hum”,“hu” or “h” is added to antibody clone designations when necessary todistinguish humanized antibodies from parental rodent antibodies. Thehumanized forms of rodent antibodies will generally comprise the sameCDR sequences of the parental rodent antibodies, although certain aminoacid substitutions may be included to increase affinity, increasestability of the humanized antibody, or for other reasons.

The term “fully human antibody” refers to an antibody that compriseshuman immunoglobulin protein sequences only. A fully human antibody maycontain murine carbohydrate chains if produced in a mouse, in a mousecell, or in a hybridoma derived from a mouse cell. Similarly, “mouseantibody” or “rat antibody” refer to an antibody that comprises onlymouse or rat immunoglobulin sequences, respectively. A fully humanantibody may be generated in a human being, in a transgenic animalhaving human immunoglobulin germline sequences, by phage display orother molecular biological methods. Exemplary techniques that can beused to make antibodies are described in U.S. Pat. Nos. 6,150,584;6,458,592; 6,420,140. Other techniques, such as the use of libraries,are known in the art.

The antibodies of the present invention also include antibodies withmodified (or blocked) Fc regions to provide altered effector functions.See, e.g., U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571;WO2006/0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Suchmodification can be used to enhance or suppress various reactions of theimmune system, with possible beneficial effects in diagnosis andtherapy. Alterations of the Fc region include amino acid changes(substitutions, deletions and insertions), glycosylation ordeglycosylation, and adding multiple Fc. Changes to the Fc can alsoalter the half-life of antibodies in therapeutic antibodies, and alonger half-life would result in less frequent dosing, with theconcomitant increased convenience and decreased use of material. SeePresta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.

The antibodies of the present invention also include antibodies withintact Fc regions that provide full effector functions, e.g. antibodiesof isotype IgG1, which induce complement-dependent cytotoxicity (CDC) orantibody dependent cellular cytotoxicity (ADCC) in a targeted cell.

The antibodies of the present invention also include antibodiesconjugated to cytotoxic payloads, such as cytotoxic agents orradionuclides. Such antibody conjugates may be used in immunotherapy inconjunction with anti-GITR, anti-PD-1, or anti PD-L1 treatment, toselectively target and kill cells expressing certain antigens on theirsurface. Exemplary cytotoxic agents include ricin, vinca alkaloid,methotrexate, Psuedomonas exotoxin, saporin, diphtheria toxin,cisplatin, doxorubicin, abrin toxin, gelonin and pokeweed antiviralprotein. Exemplary radionuclides for use in immunotherapy with theantibodies of the present invention include ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu,²¹¹At, ¹⁷⁷Lu, ¹⁴³Pr and ²¹³Bi. See, e.g., U.S. Patent ApplicationPublication No. 2006/0014225.

Bispecific antibodies are also useful in the present methods andcompositions. As used herein, the term “bispecific antibody” refers toan antibody, typically a monoclonal antibody, having bindingspecificities for at least two different antigenic epitopes. In oneembodiment, the epitopes are from the same antigen. In anotherembodiment, the epitopes are from two different antigens. Methods formaking bispecific antibodies are known in the art. For example,bispecific antibodies can be produced recombinantly using theco-expression of two immunoglobulin heavy chain/light chain pairs. See,e.g., Milstein, et al. (1983) Nature 305: 537-39. Alternatively,bispecific antibodies can be prepared using chemical linkage. See, e.g.,Brennan, et al. (1985) Science 229:81. Bispecific antibodies includebispecific antibody fragments. See, e.g., Holliger, et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90:6444-48, Gruber, et al. (1994) J. Immunol.152:5368.

The term “multispecific” includes binding molecules having specificityfor more than one target antigen. Such molecules have more than onebinding site where each binding site specifically binds (e.g.,immunoreacts with) a different target molecule or a different antigenicsite on the same target. In one embodiment, a multispecific bindingmolecule of the invention is a bispecific molecule (e.g., antibody,minibody, domain deleted antibody, or fusion protein) having bindingspecificity for at least two targets, e.g., more than one targetmolecule or more than one epitope on the same target molecule.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody that are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34(CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variabledomain and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) inthe heavy chain variable domain (Kabat et al. (1991) Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.) and/or those residues froma “hypervariable loop” (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96(L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and96-101 (H3) in the heavy chain variable domain (Chothia and Lesk (1987)J. Mol. Biol. 196: 901-917). As used herein, the term “framework” or“FR” residues refers to those variable domain residues other than thehypervariable region residues defined herein as CDR residues. Theresidue numbering above relates to the Kabat numbering system.

“Binding compound” refers to a molecule, small molecule, macromolecule,polypeptide, antibody or fragment or analogue thereof, or solublereceptor, capable of binding to a target. “Binding compound” also mayrefer to a complex of molecules, e.g., a non-covalent complex, to anionized molecule, and to a covalently or non-covalently modifiedmolecule, e.g., modified by phosphorylation, acylation, cross-linking,cyclization, or limited cleavage, that is capable of binding to atarget. When used with reference to antibodies, the term “bindingcompound” refers to both antibodies and antigen binding fragmentsthereof “Binding” refers to an association of the binding compositionwith a target where the association results in reduction in the normalBrownian motion of the binding composition, in cases where the bindingcomposition can be dissolved or suspended in solution. “Bindingcomposition” refers to a molecule, e.g. a binding compound, incombination with a stabilizer, excipient, salt, buffer, solvent, oradditive, capable of binding to a target.

As used herein, “conservatively modified variants” of or “conservativesubstitution” refers to substitutions of amino acids that are known tothose of skill in this art and may often be made even in essentialregions of the antibody without altering the biological activity of theresulting antibody. Such exemplary substitutions are preferably made inaccordance with those set forth in Table 1 as follows:

TABLE 1 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys, His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

Those of skill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide may notsubstantially alter biological activity. See, e.g., Watson, et al.(1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p.224 (4th Edition).

The phrase “consists essentially of,” or variations such as “consistessentially of” or “consisting essentially of,” as used throughout thespecification and claims, indicate the inclusion of any recited elementsor group of elements, and the optional inclusion of other elements, ofsimilar or different nature than the recited elements, that do notmaterially change the basic or novel properties of the specified dosageregimen, method, or composition. As a non-limiting example, a bindingcompound that consists essentially of a recited amino acid sequence mayalso include one or more amino acids, including substitutions of one ormore amino acid residues, that do not materially affect the propertiesof the binding compound.

“Immune condition” or “immune disorder” encompasses, e.g., pathologicalinflammation, an inflammatory disorder, and an autoimmune disorder ordisease. “Immune condition” also refers to infections, persistentinfections, and proliferative conditions, such as cancer, tumors, andangiogenesis, including infections, tumors, and cancers that resisteradication by the immune system. “Cancerous condition” includes, e.g.,cancer, cancer cells, tumors, angiogenesis, and precancerous conditionssuch as dysplasia.

“Proliferative activity” encompasses an activity that promotes, that isnecessary for, or that is specifically associated with, e.g., normalcell division, as well as cancer, tumors, dysplasia, celltransformation, metastasis, and angiogenesis.

The terms “cancer”, “tumor”, “cancerous”, and “malignant” refer to ordescribe the physiological condition in mammals that is typicallycharacterized by unregulated cell growth. Examples of cancer include butare not limited to, carcinoma including adenocarcinoma, lymphoma,blastoma, melanoma, sarcoma, and leukemia. More particular examples ofsuch cancers include squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, Hodgkin's andnon-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma,cervical cancer, ovarian cancer, liver cancer such as hepatic carcinomaand hepatoma, bladder cancer, breast cancer, colon cancer, colorectalcancer, endometrial carcinoma, myeloma (such as multiple myeloma),salivary gland carcinoma, kidney cancer such as renal cell carcinoma andWilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulvalcancer, thyroid cancer, testicular cancer, esophageal cancer, andvarious types of head and neck cancer.

As cancerous cells grow and multiply, they form a mass of canceroustissue, that is a tumor, which invades and destroys normal adjacenttissues. Malignant tumors are cancer. Malignant tumors usually can beremoved, but they may grow back. Cells from malignant tumors can invadeand damage nearby tissues and organs. Also, cancer cells can break awayfrom a malignant tumor and enter the bloodstream or lymphatic system,which is the way cancer cells spread from the primary tumor (i.e., theoriginal cancer) to form new tumors in other organs. The spread ofcancer in the body is called metastasis (What You Need to Know AboutCancer—an Overview, NIH Publication No. 00-1566; posted Sep. 26, 2000,updated Sep. 16, 2002 (2002)).

As used herein, the term “solid tumor” refers to an abnormal growth ormass of tissue that usually does not contain cysts or liquid areas.Solid tumors may be benign (not cancerous) or malignant (cancerous).Different types of solid tumors are named for the type of cells thatform them. Examples of solid tumors are sarcomas, carcinomas, andlymphomas. Leukemias (cancers of the blood) generally do not form solidtumors (National Cancer Institute, Dictionary of Cancer Terms).

“Tumor burden” also referred to as “tumor load”, refers to the totalamount of tumor material distributed throughout the body. Tumor burdenrefers to the total number of cancer cells or the total size oftumor(s), throughout the body, including lymph nodes and bone barrow.Tumor burden can be determined by a variety of methods known in the art,such as, e.g. by measuring the dimensions of tumor(s) upon removal fromthe subject, e.g., using calipers, or while in the body using imagingtechniques, e.g., ultrasound, bone scan, computed tomography (CT) ormagnetic resonance imaging (MM) scans.

The term “tumor size” refers to the total size of the tumor which can bemeasured as the length and width of a tumor. Tumor size may bedetermined by a variety of methods known in the art, such as, e.g. bymeasuring the dimensions of tumor(s) upon removal from the subject,e.g., using calipers, or while in the body using imaging techniques,e.g., bone scan, ultrasound, CT or MRI scans.

As used herein, the term “primary cancer” refers to the original tumoror the first tumor. Cancer may begin in any organ or tissue of the body.It is usually named for the part of the body or the type of cell inwhich it originates (Metastatic Cancer: Questions and Answers, CancerFacts 6.20, National Cancer Institute, reviewed Sep. 1, 2004 (2004)).

As used herein, the term “carcinoma in situ” refers to cancerous cellsthat are still contained within the tissue where they started to grow,and have not yet become invasive or spread to other parts of the body.

As used herein, the term “carcinomas” refers to cancers of epithelialcells, which are cells that cover the surface of the body, producehormones, and make up glands. Examples of carcinomas are cancers of theskin, lung, colon, stomach, breast, prostate and thyroid gland.

As used herein, the term “isolated nucleic acid molecule” refers to anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the antibody nucleic acid. An isolated nucleicacid molecule is other than in the form or setting in which it is foundin nature. Isolated nucleic acid molecules therefore are distinguishedfrom the nucleic acid molecule as it exists in natural cells. However,an isolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the antibody where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

The expression “control sequences” refers to DNA sequences involved inthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to use promoters,polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “polymerase chain reaction” or “PCR” refers to aprocedure or technique in which minute amounts of a specific piece ofnucleic acid, RNA and/or DNA, are amplified as described in, e.g., U.S.Pat. No. 4,683,195. Generally, sequence information from the ends of theregion of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to opposite strands of the template to beamplified. The 5′ terminal nucleotides of the two primers can coincidewith the ends of the amplified material. PCR can be used to amplifyspecific RNA sequences, specific DNA sequences from total genomic DNA,and cDNA transcribed from total cellular RNA, bacteriophage or plasmidsequences, etc. See generally Mullis, et al. (1987) Cold Spring HarborSymp. Quant. Biol. 51:263; Erlich, ed., (1989) PCR TECHNOLOGY (StocktonPress, N.Y.) As used herein, PCR is considered to be one, but not theonly, example of a nucleic acid polymerase reaction method foramplifying a nucleic acid test sample comprising the use of a knownnucleic acid as a primer and a nucleic acid polymerase to amplify orgenerate a specific piece of nucleic acid.

As used herein, the term “germline sequence” refers to a sequence ofunrearranged immunoglobulin DNA sequences, including rodent (e.g. mouse)and human germline sequences. Any suitable source of unrearrangedimmunoglobulin DNA may be used. Human germline sequences may beobtained, for example, from JOINSOLVER® germline databases on thewebsite for the National Institute of Arthritis and Musculoskeletal andSkin Diseases of the United States National Institutes of Health. Mousegermline sequences may be obtained, for example, as described inGiudicelli et al. (2005) Nucleic Acids Res. 33:D256-D261.

To examine the extent of enhancement of, e.g., GITR activity, samples orassays comprising a given, e.g., protein, gene, cell, or organism, aretreated with a potential activating or inhibiting agent and are comparedto control samples treated with an inactive control molecule. Controlsamples are assigned a relative activity value of 100%. Inhibition isachieved when the activity value relative to the control is about 90% orless, typically 85% or less, more typically 80% or less, most typically75% or less, generally 70% or less, more generally 65% or less, mostgenerally 60% or less, typically 55% or less, usually 50% or less, moreusually 45% or less, most usually 40% or less, preferably 35% or less,more preferably 30% or less, still more preferably 25% or less, and mostpreferably less than 20%. Activation is achieved when the activity valuerelative to the control is about 110%, generally at least 120%, moregenerally at least 140%, more generally at least 160%, often at least180%, more often at least 2-fold, most often at least 2.5-fold, usuallyat least 5-fold, more usually at least 10-fold, preferably at least20-fold, more preferably at least 40-fold, and most preferably over40-fold higher.

Endpoints in activation or inhibition can be monitored as follows.Activation, inhibition, and response to treatment, e.g., of a cell,physiological fluid, tissue, organ, and animal or human subject, can bemonitored by an endpoint. The endpoint may comprise a predeterminedquantity or percentage of, e.g., an indicia of inflammation,oncogenicity, or cell degranulation or secretion, such as the release ofa cytokine, toxic oxygen, or a protease. The endpoint may comprise,e.g., a predetermined quantity of ion flux or transport; cell migration;cell adhesion; cell proliferation; potential for metastasis; celldifferentiation; and change in phenotype, e.g., change in expression ofgene relating to inflammation, apoptosis, transformation, cell cycle, ormetastasis (see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30:145-158;Hood and Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme, et al.(2003) Curr. Drug Targets 4:251-261; Robbins and Itzkowitz (2002) Med.Clin. North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Rev.Genomics Hum. Genet. 3:101-128; Bauer, et al. (2001) Glia 36:235-243;Stanimirovic and Satoh (2000) Brain Pathol. 10:113-126).

An endpoint of inhibition is generally 75% of the control or less,preferably 50% of the control or less, more preferably 25% of thecontrol or less, and most preferably 10% of the control or less.Generally, an endpoint of activation is at least 150% the control,preferably at least two times the control, more preferably at least fourtimes the control, and most preferably at least 10 times the control.

“Small molecule” is defined as a molecule with a molecular weight thatis less than 10 kDa, typically less than 2 kDa, and preferably less than1 kDa. Small molecules include, but are not limited to, inorganicmolecules, organic molecules, organic molecules containing an inorganiccomponent, molecules comprising a radioactive atom, synthetic molecules,peptide mimetics, and antibody mimetics. As a therapeutic, a smallmolecule may be more permeable to cells, less susceptible todegradation, and less apt to elicit an immune response than largemolecules. Small molecules, such as peptide mimetics of antibodies andcytokines, as well as small molecule toxins are described. See, e.g.,Casset, et al. (2003) Biochem. Biophys. Res. Commun. 307:198-205;Muyldermans (2001) J. Biotechnol. 74:277-302; Li (2000) Nat. Biotechnol.18:1251-1256; Apostolopoulos, et al. (2002) Curr. Med. Chem. 9:411-420;Monfardini, et al. (2002) Curr. Pharm. Des. 8:2185-2199; Domingues, etal. (1999) Nat. Struct. Biol. 6:652-656; Sato and Sone (2003) Biochem.J. 371:603-608; U.S. Pat. No. 6,326,482.

“Specifically” or “selectively” binds, when referring to aligand/receptor, antibody/antigen, or other binding pair, indicates abinding reaction that is determinative of the presence of the protein ina heterogeneous population of proteins and other biologics. Thus, underdesignated conditions, a specified ligand binds to a particular receptorand does not bind in a significant amount to other proteins present inthe sample. As used herein, an antibody is said to bind specifically toa polypeptide comprising a given sequence (in this case GITR) if itbinds to polypeptides comprising the sequence of GITR but does not bindto proteins lacking the sequence of GITR. For example, an antibody thatspecifically binds to a polypeptide comprising GITR may bind to aFLAG®-tagged form of GITR but will not bind to other FLAG®-taggedproteins.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which a binding molecule specifically binds. Epitopes can beformed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in aunique spatial conformation. Methods of determining spatial conformationof epitopes include, for example, X-ray crystallography and2-dimensional nuclear magnetic resonance.

Binding molecules that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen, i.e., a competitivebinding assay. Competitive binding is determined in an assay in whichthe binding molecule being tested inhibits specific binding of areference binding molecule to a common antigen, such as GITR. Numeroustypes of competitive binding assays are known, for example: solid phasedirect or indirect radioimmunoassay (RIA); solid phase direct orindirect enzyme immunoassay (EIA) sandwich competition assay (seeStahli, et al., (1983) Methods in Enzymology 9:242); solid phase directbiotin-avidin EIA (see Kirkland, et al., (1986) J. Immunol. 137:3614);solid phase direct labeled assay, solid phase direct labeled sandwichassay (see Harlow and Lane, (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Press); solid phase direct label RIA using 1-125 label(see Morel, et al., (1988) Mol. Immunol. 25(1):7); solid phase directbiotin-avidin EIA (Cheung, et al., (1990) Virology 176:546); and directlabeled RIA. (Moldenhauer, et al., (1990) Scand. J. Immunol. 32:77).

Typically, such an assay involves the use of purified antigen bound to asolid surface or cells bearing either of these, an unlabeled testbinding molecule and a labeled reference binding molecule. Competitiveinhibition is measured by determining the amount of label bound to thesolid surface or cells in the presence of the test binding molecule.

Usually the test binding molecule is present in excess. Usually, when acompeting binding molecule is present in excess, it will inhibitspecific binding of a reference binding molecule to a common antigen byat least 50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.

The antibody, or binding composition derived from the antigen-bindingsite of an antibody, of the contemplated method binds to its antigenwith an affinity that is at least two fold greater, preferably at leastten times greater, more preferably at least 20-times greater, and mostpreferably at least 100-times greater than the affinity with unrelatedantigens. In a preferred embodiment the antibody will have an affinitythat is greater than about 10⁹ liters/mol, as determined, e.g., byScatchard analysis. Munsen, et al. (1980) Analyt. Biochem. 107:220-239.

II. General

The present invention provides methods of treating advanced stage tumorswith a combination of GITR agonists and PD-1 antagonists, includinganti-GITR and anti-PD-1 or anti-PD-L1 antibodies.

III. Pharmaceutical Compositions

To prepare pharmaceutical or sterile compositions, the GITR, PD-1, orPD-L1 antibodies are admixed with a pharmaceutically acceptable carrieror excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S.Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa.(1984).

Formulations of therapeutic and diagnostic agents may be prepared bymixing with physiologically acceptable carriers, excipients, orstabilizers in the form of, e.g., lyophilized powders, slurries, aqueoussolutions or suspensions. See, e.g., Hardman et al. (2001) Goodman andGilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, NewYork, N.Y.; Gennaro (2000) Remington: The Science and Practice ofPharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, etal. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications,Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.

Toxicity and therapeutic efficacy of the antibody compositions,administered alone or in combination with an immunosuppressive agent,can be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio of LD₅₀ to ED₅₀. Antibodies exhibiting high therapeuticindices are preferred. The data obtained from these cell culture assaysand animal studies can be used in formulating a range of dosage for usein human. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration.

The mode of administration is not particularly important. Suitableroutes of administration may, for example, include oral, rectal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. Administrationof antibody used in the pharmaceutical composition or to practice themethod of the present invention can be carried out in a variety ofconventional ways, such as oral ingestion, inhalation, topicalapplication or cutaneous, subcutaneous, intraperitoneal, parenteral,intraarterial or intravenous injection.

Alternately, one may administer the antibody in a local rather thansystemic manner, for example, via injection of the antibody directlyinto an arthritic joint or pathogen-induced lesion characterized byimmunopathology, often in a depot or sustained release formulation.Furthermore, one may administer the antibody in a targeted drug deliverysystem, for example, in a liposome coated with a tissue-specificantibody, targeting, for example, arthritic joint or pathogen-inducedlesion characterized by immunopathology. The liposomes will be targetedto and taken up selectively by the afflicted tissue.

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. Preferably,an administration regimen maximizes the amount of therapeutic deliveredto the patient consistent with an acceptable level of side effects.Accordingly, the amount of biologic delivered depends in part on theparticular entity and the severity of the condition being treated.Guidance in selecting appropriate doses of antibodies, cytokines, andsmall molecules are available. See, e.g., Wawrzynczak (1996) AntibodyTherapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991)Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York,N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy inAutoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003)New Engl. J. Med. 348:601-608; Milgrom, et al. (1999) New Engl. J. Med.341:1966-1973; Slamon, et al. (2001) New Engl. J. Med. 344:783-792;Beniaminovitz, et al. (2000) New Engl. J. Med. 342:613-619; Ghosh, etal. (2003) New Engl. J. Med. 348:24-32; Lipsky, et al. (2000) New Engl.J. Med. 343:1594-1602.

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced. Preferably, a biologic that will beused is substantially derived from the same species as the animaltargeted for treatment (e.g. a humanized antibody for treatment of humansubjects), thereby minimizing any immune response to the reagent.

Antibodies and antibody fragments can be provided by continuousinfusion, or by doses at intervals of, e.g., one day, 1-7 times perweek, one week, two weeks, monthly, bimonthly, etc. Doses may beprovided intravenously, subcutaneously, topically, orally, nasally,rectally, intramuscular, intracerebrally, intraspinally, or byinhalation. A preferred dose protocol is one involving the maximal doseor dose frequency that avoids significant undesirable side effects. Atotal weekly dose is generally at least 0.05 μg/kg, 0.2 μg/kg, 0.5μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.2 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10mg/kg, 25 mg/kg, 50 mg/kg body weight or more. See, e.g., Yang, et al.(2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J.Med. 346:1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych.67:451-456; Portielji, et al. (20003) Cancer Immunol. Immunother.52:133-144. The desired dose of a small molecule therapeutic, e.g., apeptide mimetic, natural product, or organic chemical, is about the sameas for an antibody or polypeptide, on a moles/kg basis.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, antibody, steroid, chemotherapeutic agent,antibiotic, anti-viral, or radiation, are well known in the art, see,e.g., Hardman, et al. (eds.) (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York,N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for AdvancedPractice: A Practical Approach, Lippincott, Williams & Wilkins, Phila.,Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy,Lippincott, Williams & Wilkins, Phila., Pa. In particular,administration of PD-1 or PD-L1 antibodies can occur simultaneously orsequentially. In particular embodiments, the anti-GITR antibody can beadministered first followed by periodic (e.g. one week later or weekly)dosing of an anti-PD-1, or anti-PD-L1 antibodies. Alternatively,treatment with anti-PD-1 or PD-L1 antibodies can be followed bytreatment with anti-GITR antibodies on a similar schedule. In furtherembodiments, anti-GITR antibodies are co-administered with anti-PD-1 oranti-PD-L1 in at least a single treatment or multiple doses (e.g.,weekly administration).

The GITR, PD-1 or PD-L1 antibodies can be combined with chemotherapeuticagents including alkylating agents such as thiotepa and CYTOXAN®cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; XELODA® capecitabine; ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such asretinoic acid; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens and selective estrogenreceptor modulators (SERMs), including, for example, tamoxifen(including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON. toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A combination therapy is used to treat an advanced stage tumor havingdimensions of at least about 175 mm³. In another embodiment of theinvention, a combination therapy is used to treat a tumor that is atleast about 200 mm³, 300 mm³, 400 mm³, 500 mm³, 750 mm³, up to 1000 mm³.A combination therapy of the invention is used to treat a tumor that islarge enough to be found by palpation or by imaging techniques wellknown in the art, such as MRI, ultrasound, or CAT scan.

A “synergistic effect” of two compounds is one in which the effect ofthe combination of the two agents is greater than the sum of theirindividual effects and is statistically different from the controls andthe single drugs. In another embodiment, the combination therapies ofthe invention have an additive effect. An “additive effect” of twocompounds is one in which the effect of the combination of the twoagents is the sum of their individual effects and is statisticallydifferent from either the controls and/or the single drugs.

The subject methods result in an inhibition of tumor size more thanabout 10%, more than about 20%, more than about 30%, more than about35%, more than about 42%, more than about 43%, more than about 44%, morethan about 45%, more than about 46%, more than about 47%, more thanabout 48%, more than about 49%, more than about 50%, more than about51%, more than about 52%, more than about 53%, more than about 54%, morethan about 55%, more than about 56%, more than about 57%, more thanabout 58%, more than about 59%, more than about 60%, more than about65%, more than about 70%, more than about 75%, more than about 80%, morethan about 85%, more than about 90%, more than about 95%, or more thanabout 100%. In one embodiment, the administration of a GITR bindingmolecule in conjunction with a PD-1 antagonist molecule can lead tocomplete regression of an advanced tumor.

Also contemplated is co-administration of the GITR agonist/PD-1antagonist combination with anti-viral therapeutics. Anti-virals includeany drug that destroys viruses. Antivirals may include interferons,which function to inhibit replication of the virus, protease inhibitors,and reverse transcriptase inhibitors or agents contained in thecombination of highly active antiretroviral therapy (HAART) for HIV.

Typical veterinary, experimental, or research subjects include monkeys,dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.

IV. Uses Cancer

The GITR, PD-1, or PD-L1 antibodies or antigen binding fragments can beused to treat cancer (i.e., to inhibit the growth or survival of tumorcells). Preferred cancers whose growth may be inhibited using theantibodies of the invention include cancers typically responsive toimmunotherapy, but also cancers that have not hitherto been associatedwith immunotherapy. Non-limiting examples of preferred cancers fortreatment include melanoma (e.g., metastatic malignant melanoma), renalcancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormonerefractory prostate adenocarcinoma), pancreatic adenocarcinoma, breastcancer, colon cancer, lung cancer (e.g. non-small cell lung cancer),esophageal cancer, squamous cell carcinoma of the head and neck, livercancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma,glioma, leukemia, lymphoma, and other neoplastic malignancies.Additionally, the invention includes refractory or recurrentmalignancies whose growth may be inhibited using the antibodies of theinvention.

The GITR agonist/PD-1 antagonist antibody or antigen binding fragmentscan be used alone or in combination with: other anti-neoplastic agentsor immunogenic agents (for example, attenuated cancerous cells, tumorantigens (including recombinant proteins, peptides, and carbohydratemolecules), antigen presenting cells such as dendritic cells pulsed withtumor derived antigen or nucleic acids, immune stimulating cytokines(for example, IL-2, IFNa2, GM-CSF), and cells transfected with genesencoding immune stimulating cytokines such as but not limited toGM-CSF); standard cancer treatments (for example, chemotherapy,radiotherapy or surgery); or other antibodies (including but not limitedto antibodies to VEGF, EGFR, Her2/neu, VEGF receptors, other growthfactor receptors, CD20, CD40, CTLA-4, OX-40, 4-IBB, and ICOS).

Infectious Diseases

The GITR agonist/PD-1 antagonist combination can also be used to preventor treat infections and infectious disease. The GITR agonist/PD-1antagonist combination can be used alone, or in conjunction withvaccines, to stimulate the immune response to pathogens, toxins, andself-antigens. The antibodies or antigen-binding fragment thereof can beused to stimulate immune response to viruses infectious to humans, suchas, but not limited to, human immunodeficiency viruses, hepatitisviruses class A, B and C, Eppstein Barr virus, human cytomegalovirus,human papilloma viruses, herpes viruses. The antibodies orantigen-binding fragment thereof can be used to stimulate immuneresponse to infection with bacterial or fungal parasites, and otherpathogens.

Vaccination Adjuvants

The GITR agonist/PD-1 antagonist antibody or antibody fragmentcombination can be used in conjunction with other recombinant proteinsand/or peptides (such as tumor antigens or cancer cells) in order toincrease an immune response to these proteins (i.e., in a vaccinationprotocol).

For example, GITR agonist/PD-1 antagonist antibodies and antibodyfragments thereof may be used to stimulate antigen-specific immuneresponses by co-administration of the GITR agonist/PD-1 antagonistcombination with an antigen of interest (e.g., a vaccine). Accordingly,in another aspect the invention provides a method of enhancing an immuneresponse to an antigen in a subject, comprising administering to thesubject: (i) the antigen; and (ii) a GITR agonist/PD-1 antagonistcombination, such that an immune response to the antigen in the subjectis enhanced. The antigen can be, for example, a tumor antigen, a viralantigen, a bacterial antigen or an antigen from a pathogen.

Ex-Vivo Activation of T Cells

The antibodies and antigen fragments of the invention can also be usedfor the ex vivo activation and expansion of antigen specific T cells andadoptive transfer of these cells into recipients in order to increaseantigen-specific T cells against tumor. These methods may also be usedto activate T cell responses to infectious agents such as CMV. Ex vivoactivation in the presence of the GITR agonist/PD-1 antagonistcombination may be expected to increase the frequency and activity ofthe adoptively transferred T cells.

EXAMPLES Example 1 General Methods

Standard methods in molecular biology are described. Maniatis, et al.(1982) Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001)Molecular Cloning, 3^(rd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, AcademicPress, San Diego, Calif. Standard methods also appear in Ausbel, et al.(2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley andSons, Inc. New York, N.Y., which describes cloning in bacterial cellsand DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol.2), glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed. Coligan, et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemicalmodification, post-translational modification, production of fusionproteins, glycosylation of proteins are described. See, e.g., Coligan,et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wileyand Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols inMolecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp.16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life ScienceResearch, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001)BioDirectory, Piscataway, N.J., pp. 384-391. Production, purification,and fragmentation of polyclonal and monoclonal antibodies are described.Coligan, et al. (2001) Current Protcols in Immunology, Vol. 1, JohnWiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlowand Lane, supra. Standard techniques for characterizing ligand/receptorinteractions are available. See, e.g., Coligan, et al. (2001) CurrentProtcols in Immunology, Vol. 4, John Wiley, Inc., New York.

Monoclonal, polyclonal, and humanized antibodies can be prepared (see,e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ.Press, New York, N.Y.; Kontermann and Dubel (eds.) (2001) AntibodyEngineering, Springer-Verlag, New York; Harlow and Lane (1988)Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp. 139-243; Carpenter, et al. (2000) J.Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang, et al.(1999) J. Biol. Chem. 274:27371-27378; Baca, et al. (1997) J. Biol.Chem. 272:10678-10684; Chothia, et al. (1989) Nature 342:877-883; Footeand Winter (1992)J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

An alternative to humanization is to use human antibody librariesdisplayed on phage or human antibody libraries in transgenic mice(Vaughan, et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995)Nature Medicine 1:837-839; Mendez, et al. (1997) Nature Genetics15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377;Barbas, et al. (2001) Phage Display: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Kay, et al. (1996)Phage Display of Peptides and Proteins: A Laboratory Manual, AcademicPress, San Diego, Calif.; de Bruin, et al. (1999) Nature Biotechnol.17:397-399).

Purification of antigen is not necessary for the generation ofantibodies. Animals can be immunized with cells bearing the antigen ofinterest. Splenocytes can then be isolated from the immunized animals,and the splenocytes can fused with a myeloma cell line to produce ahybridoma (see, e.g., Meyaard, et al. (1997) Immunity 7:283-290; Wright,et al. (2000) Immunity 13:233-242; Preston, et al., supra; Kaithamana,et al. (1999) J. Immunol. 163:5157-5164).

Antibodies can be conjugated, e.g., to small drug molecules, enzymes,liposomes, polyethylene glycol (PEG). Antibodies are useful fortherapeutic, diagnostic, kit or other purposes, and include antibodiescoupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g.,colloidal gold (see, e.g., Le Doussal, et al. (1991) J. Immunol.146:169-175; Gibellini, et al. (1998) J. Immunol. 160:3891-3898; Hsingand Bishop (1999) J. Immunol. 162:2804-2811; Everts, et al. (2002) J.Immunol. 168:883-889).

Methods for flow cytometry, including fluorescence activated cellsorting detection systems (FACS®), are available. See, e.g., Owens etal. (1994) Flow Cytometry Principles for Clinical Laboratory Practice,John Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd)ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry,John Wiley and Sons, Hoboken, N.J. Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable. Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

Standard methods of histology of the immune system are described. See,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available. See, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne et al. (2000) Bioinformatics 16: 741-742;Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren etal. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690.

Example 2 In Vivo Treatment Methods

Approximately eight to ten week old female C57Bl/6J or BALB/c/AnN micewere obtained from Jackson Laboratories (Bar Harbor, Me. or Sacramento,Calif.) or Taconic Laboratory (Oxnard, Calif.), respectively.Conventional animal chow and water were provided ad libitum. Allprotocols using animals have been approved by Merck & Co., Inc. andMerck Research Labs (MRL) Palo Alto Animal Use and Care Committee.

Before treatment, mice were weighed and tumors from individual mice weremeasured. To prevent bias, any outliers by weight or tumor volume wereremoved and the remaining mice randomized into various treatment groupswith equivalent mean tumor size.

The test materials and isotype controls were obtained from MRL Palo AltoProtein Sciences department as frozen (−80° C.) stocks. The formulationbuffers were specific for each antibody to stabilize proteins andprevent precipitation, the details of which are given below:

The formulations/diluents were obtained from MRL Palo Alto ProteinSciences department as stored at 4° C. The isotype control mIgG2a andanti-PD-1 formulation/diluent of 20 mM Na Acetate, 7% sucrose, pH5.5,mIgG1 formulation/diluent of 75 mM NaCl, 10 mM Phosphate, 3% sucrose,pH7.3, and the mDTA-1 (anti-mGITR) formulation/diluent of 20 mMNaAcetate, 7% sucrose, 0.02% Tween80 low peroxide, pH5.5 were forstabilizing the proteins and preventing from precipitation.

Example 3 Tumor Cell Line Preparation and Implant

MC38 or CT26 colon carcinoma cells were cultured in RPMI mediumsupplemented with 10% heat-inactivated fetal bovine serum. 1×1 MB49bladder carcinoma cells were cultured in Dulbecco's Modified EagleMedium (DMEM) supplemented with 10% fetal bovine serum and 1% GlutaMAX™.1×10⁶ cells of MC38, 3×10⁵ cells of CT26, or 0.5×10⁶ cells of MB49 cellswere injected SC in 100 μL volume of phosphate buffered saline in theleft belly area or right flank of each mouse. Typically mice were firstshaved with electronic clippers in the area that would be used for theimplant.

Example 4 Tumor Measurements and Body Weights

Tumors were measured the day before the first dose and twice a weekthereafter. Tumor length and width were measured using electroniccalipers and tumor volume determined using the formula Volume(mm³)=0.5×Length×Width where length is the longer dimension.

Mice were weighed periodically to monitor general health but also toestimate actual mg/kg dose delivery per mouse where needed.

Example 5 Dosing Solution Preparation, Administration, and Analyses

Frozen stocks were thawed and transferred to wet ice. To avoid repeatedfreeze thaw, each vial of stock was thawed once and aliquots made involumes sufficient for one time use. Polypropylene, low adhesion tubeswere used for this purpose. The aliquots were snap frozen in dry ice andstored at 80° C. Before each dosing, one aliquot was thawed and dilutedto nominal concentration in the appropriate diluent and dosedimmediately. Aliquots of dosing solutions were snap frozen in dry iceand stored at −80° C. until analyses. Dosing solutions were assessedusing the Meso Scale Discovery (MSD®, Rockville, Md.) platform which isbased on multi-array technology; a combination ofelectrochemiluminescence detection and patterned arrays.

Dosing with the test materials started once the MC38 and CT26 tumorsreached an average size of approximately 300 mm³ and 220 mm³,respectively, typically around two weeks post implant. Dosing with thetest materials started once the MB49 tumors reached an average size ofapproximately 105 mm³, one week post implant. Variations in dosingfrequency (ranging from a single dose to up to 6 weekly doses) at adosing concentration of 5 mg/kg were tested, the details of which aregiven in below.

Example 6 Murinization of DTA-1 Antibody

Rat anti-mouse DTA-1 GITR antibody (S. Sakaguchi, Kyoto University,Kyoto, Japan), was murinized as follows. The sequence of rat antibodyDTA-1 was determined for the variable heavy (VH) and variable light (VL)domains. The rat DTA-1 VH sequence was compared to mouse VH germlinesequences from the Immunogenetics IMGT Database (www.imgt.org) (Lefranc,M.-P. et al. (1999) Nuc. Acids Res. 27:209-212). DTA-1 VH sequence wasaligned with the mouse VH germline sequences and scored similarly to aprevious humanization system (see, e.g. WO 2005/047326). The rat DTA-1VH was most similar to mouse germlines IGVH5-4, IGVH5-6 and IGVH5-9. CDRresidues were transferred from rat DTA-1 VH to mouse germline IGVH5-4;two IGVH5-4 framework residues were altered to those fitting IGVH5-6 andmouse J-region IGHJ-4 (IMGT) was used to connect to mouse IgG1 and mouseIgG2a Fc regions.

The rat DTA-1 VL (lambda) sequence was aligned with mouse VL (lambda)sequence from GenBank: AAH02129.1. CDR residues were transferred fromrat DTA-1 VL (lambda) to the mouse AAH02129 framework sequence. Sevenframework residues on murinized DTA1 were altered based on computergraphic models of the rat and murinized VL domains. The murinized DTA-1VL (lambda) domain was fused to mouse constant light domain.

For all three constructs (one VH and two VL (lambda)), codon-optimizedgenes were synthesized and inserted into expression vectors. Antibodieswere expressed by transient expression in HEK293 cells and purifiedusing protein-A chromatography.

Example 7 Anti-GITR/Anti-PD-1 Treatment Results

Advanced MC38 tumor-bearing C57BL/6J mice were treated with a single ortwo weekly injections of murinized anti-mGITR (Merck Research Labs, PaloAlto, Calif.) subcutaneously (SC), and murinized anti-mPD-1 (MerckResearch Labs, Palo Alto, Calif.) intraperitoneally (IP), dosed at 5mg/kg each. Treatment was started once the tumor sizes reached 240-360mm³. Tumors were measured twice weekly. Complete regression (CR) oftumors served as a read-out for anti-tumor efficacy. Combination dosingled to robust, synergistic efficacy, with 100% CR after two weeklycombination doses. Limiting the regimen to one dose of each antibody(Ab.) reduced CR to 70%, similar to that achieved with combination ofanti-mGITR followed by four weekly administrations of anti-mPD-1beginning one week later. A two week interval between anti-mGITR andanti-mPD-1 administration was not as effective. Only 20-30% CR was seenwith monotherapy of up to six weekly treatments with anti-mGITR or 2-4weekly treatments of anti-mPD-1 Ab (See FIGS. 1A-1K).

Advanced MC38 tumor-bearing C57BL/6J mice were treated with anti-mGITR(SC) and anti-mPD-1 (IP) dosed at 5 mg/kg each. Treatment was startedonce the tumor size reached 200-350 mm³. Tumors were measured twiceweekly. Complete regression (CR) of tumors served as a read-out foranti-tumor efficacy. Combination dosing led to robust, synergisticefficacy, with 100% CR after two weekly combination doses. This iscomparable to the results detailed above. However, a reduced CR of 60%was observed when the antibodies were delivered separately with a oneweek interval. Two weekly monotherapy doses of either anti-mGITR oranti-mPD-1 inhibited tumor growth but did not result in CRs (see FIGS.2A-2F).

Advanced CT26 tumor-bearing BALB/cAnN mice were treated with anti-mGITR(SC) and anti-mPD-1 (IP) dosed at 5 mg/kg each. Treatment was startedonce the tumor size averaged 220 mm³ (180-260 mm³). Tumors were measuredtwice weekly. Complete regression (CR) of tumors served as a read-outfor anti-tumor efficacy. A single combination dosing led to robust,synergistic efficacy, with 70% CR. The anti-tumor efficacy with eitherantibody delivered as monotherapy was 0-10% CR (see FIGS. 3A-3D).

MB49 tumor-bearing C57BL/6J mice were treated with a single dose ofanti-GITR (SC) and anti-PD-1 (IP) at 5 mg/kg and 10 mg/kg respectively.Treatment was started once the tumor size averaged 105 mm³ (85-122 mm³).Tumors were measured twice weekly. Complete regression (CR) of tumorsserved as a read-out for anti-tumor efficacy. Anti-GITR and anti-PD-1combination treatment led to enhanced efficacy with 40% CR. No CRs wereobserved in the single agent treatment groups (see FIGS. 4A-4D).

Example 8 Effect of Anti-PD-1 and Anti-GITR Combination on Regulatory TCell and CD8 Cell Ratios A. Methods

1. Mixed Lymphocyte Reaction Cultures

Peripheral blood mononuclear cells (PBMC) were isolated from buffy coatsusing Ficoll-Paque Plus density gradient centrifugation at 1200×g for 20minutes. Peripheral blood mononuclear cells were collected from themedium:plasma interface and washed 2 times with Dulbecco'sphosphate-buffered saline (DPBS). The residual red blood cells (RBCs)were lysed using Ammonium-chloride-potassium RBC lysing solution (RBClysing solution).

Dendritic cells (DC) were generated from CD14+ monocytes using thefollowing procedure. Monocytes were first isolated from buffy coatsusing RosetteSep human monocyte enrichment cocktail and Ficoll-PaquePlus density gradient centrifugation at 1200×g for 20 minutes. Monocyteswere removed from the medium:plasma interface and washed 2 times withDPBS. The residual RBCs were lysed using RBC lysing solution. Theenriched monocytes were cultured in Dulbecco's Modified Eagle Mediumsupplemented with 10% fetal bovine serum (FBS), 1,000 U/mLgranulocyte-macrophage colony-stimulating factor (GM-CSF), and 400 U/mLinterleukin (IL)-4 at a cell density of 2×10⁶/mL. At Day 6, 0.5 μg/mLlipopolysaccharide was added to the culture; the cells were thencultured for 2 more days.

Mixed lymphocyte reaction cultures were set up in 24-well plates.Peripheral blood mononuclear cells (2×10⁶/mL) were cultured withγ-irradiated (30 Gy) allogeneic DC (0.2×10⁶/mL) in the presence of 100U/mL IL-2; 5 ng/mL IL-15; anti-hGITR antibody (MK-4166), anti-hPD-1antibody (MK-3475), combination of MK-3475 and MK-4166 or isotypecontrol mAb (anti-RSV). The number of regulatory T cells (Tregs) in MLRcultures was evaluated at Day 7 using flow cytometry.

2. Flow Cytometric Detection of T Regs in Mixed Lymphocyte ReactionCultures

For the detection of Tregs (CD3+ CD4+ CD25+ FoxP3+) and CD8+ T cells, 1to 2×10⁶ cells from MLR cultures were incubated with anti-CD3, anti-CD4,anti-CD25, and anti-CD8 in 50 μL of BD Pharmingen staining buffer. Deadcells were excluded using the Fixable Viability Dye eFluor 506. Aftersurface staining with anti-CD3, anti-CD4, anti-CD8 and anti-CD25 mAbs,intracellular FoxP3 staining was performed using the FoxP3Fixation/Permeabilization kit according to the manufacturer'sinstructions (eBioscience). Sample acquisition was performed on an LSRII flow cytometer and the data were analyzed using FlowJo softwareversion 10.0.6 (Tree Star, Inc.). Tregs were identified by gating onCD3+CD4+ cells followed by gating on CD25+ FoxP3+ cells.

3. Regulatory T-Cell Suppression Assay

CD4+ T cells were isolated from buffy coats using RosetteSep human CD4+T cells enrichment kit and Ficoll-Paque Plus density gradientcentrifugation at 1200×g for 20 minutes. CD4+ T cells were collectedfrom the medium:plasma interface and washed 2 times with DPBS. Theresidual RBCs were lysed using RBC lysing solution. CD4+ CD25+ Tregs andCD4+ CD25− effector T cells (Teffs) were separated using humanCD25-conjugated microbeads II kit according to the manufacturer'sinstructions (Miltenyi Biotec). The purity of CD4+ CD25+ CD127− Tregswas approximately 40% to 70%. Human DCs were generated as describedabove.

For the T-cell suppression assays, a total 1×10⁵ T cells (Tregs andTeffs) and 2×10⁴ γ-irradiated (30 Gy) DCs per well were cultured in96-well round-bottom plates for 7 days in the presence of MK-4166,MK-3475, combination of MK-3475 and MK-4166 or isotype control mAb(anti-RSV). CD4+ CD25− Teffs and CD4+ CD25+ Tregs were mixed at 4:1ratio. On Day 6, tritium-labeled thymidine was added to the cultures for20 hours. Following incubation with tritium-labeled thymidine, the cellswere harvested, lysed using water, and analyzed using a β counter(PerkinElmer, 2450 microplate counter). The level of T-cellproliferation was reflected by the levels of incorporatedtritium-labeled thymidine. All assays were conducted in triplicates.

B. Results

The ability of the anti-mouse GITR agonistic mAb DTA-1 to alter thestability and intratumoral accumulation of Tregs is essential for themechanism of action of DTA-1 (see, e.g., Cohen et al (2010) PLoS One5(e10436): 1-12; and Schaer et al. (2013) Cancer Immunol. Res.1:320-331). The ability of MK-4166 alone or in combination with MK-3475to impact the induction of human Tregs and their suppressive activitywas investigated using human in vitro assay.

Induction of Tregs in MLR is well documented (see, e.g. Levitsky et al(2013) Transplantation 96:689-696). Thus, MLR was utilized to increasethe number of human Tregs naturally occurring in blood and to assess theeffect of MK-4166 alone or in combination with MK-3475 on human Tregsand CD8:Treg ratio. Addition of 10 μg/mL MK-4166 to MLR culturesresulted in decreased numbers of CD4+ CD25+ FoxP3+ Tregs after 7 days(FIG. 5A). MK-3475 alone did not have an effect on the number of Tregs.However combination of MK-3475 and MK-4166 had the most pronouncedeffect on the number of Tregs and CD8:Treg ratio (FIG. 5B).

To evaluate the effect of MK-4166 on suppressive activity of humanTregs, the Treg suppression assay was established. In this assay, thelevel of T-cell proliferation is reflected by the levels of incorporatedtritium-labeled thymidine. Dose-dependent increase in T cellproliferation was observed when MK-4166 was combined with MK-3475 (FIG.6). These results provide evidence that incubation with MK-4166 andMK3475 decreases the number of MLR-induced Tregs, increases CD8:Tregration and diminishes suppressive function of human Tregs in vitro.

Table 2 provides a brief description of the sequences in the sequencelisting

SEQ ID NO: Description 1 36E5 CDRH1 2 3D6 CDRH1 3 61G6 CDRH1 4 6H6 CDRH15 61F6 CDRH1 6 1D8 CDRH1 7 17F10 CDRH1 8 35D8 CDRH1 9 49A1 CDRH1 10 9E5CDRH1 11 31H6 CDRH1 12 36E5 CDRH2 13 3D6 CDRH2 14 61G6 CDRH2 15 6H6CDRH2 16 61F6 CDRH2 17 1D8 CDRH2 18 17F10 CDRH2 19 35D8 CDRH2 20 49A1CDRH2 21 9E5 CDRH2 22 31H6 CDRH2 23 36E5 CDRH3 24 3D6 CDRH3 25 61G6CDRH3 26 6H6 CDRH3 27 61F6 CDRH3 28 1D8 CDRH3 29 17F10 CDRH3 30 35D8CDRH3 31 49A1 CDRH3 32 9E5 CDRH3 33 31H6 CDRH3 34 36E5 CDRL1 35 3D6CDRL1 36 61G6 CDRL1 37 6H6 CDRL1 38 61F6 CDRL1 39 1D8 CDRL1 40 17F10 CDRL1 41 35D8 CDR L1 42 49A1 CDR L1 43 9E5 CDR L1 44 31H6 CDR L1 45 36E5CDRL2 46 3D6 CDRL2 47 61G6 CDRL2 48 6H6 CDRL2 49 61F6 CDRL2 50 1D8 CDRL251 17F10 CDR L2 52 35D8 CDR L2 53 49A1 CDR L2 54 9E5 CDR L2 55 31H6 CDRL2 56 36E5 CDRL3 57 3D6 CDRL3 58 61G6 CDRL3 59 6H6 CDRL3 60 61F6 CDRL361 1D8 CDRL3 62 17F10 CDR L3 63 35D8 CDR L3 64 49A1 CDR L3 65 9E5 CDR L366 31H6 CDR L3 67 Humanized 1D8 VH 68 Humanized 1D8 VL 69 Humanized 3D6VH 70 Humanized 3D6 VL 71 Humanized 6H6 VH 72 Humanized 6H6 VL 73Humanized 9E5 VH 74 Humanized 9E5 VL 75 Humanized 31H6 VH 76 Humanized31H6 VL 77 Humanized 17F10 VH 78 Humanized 17F10 VL 79 Humanized 35D8 VH80 Humanized 35D8 VL 81 Humanized 36E5 VH 82 Humanized 36E5 VL 83Humanized 49A1 VH 84 Humanized 49A1 VL 85 Humanized 61F6 VH 86 Humanized61F6 VL 87 Humanized 61G6 VH 88 Humanized 61G6 VL

1. A method of treating a tumor in a patient comprising administering tothe patient a PD-1 antagonist and a GITR agonist, wherein the PD-1agonist and GITR agonist are administered simultaneously orsequentially.
 2. The method of claim 1, wherein a. the PD-1 antagonistis an antibody or antigen binding fragment thereof, that binds PD-1 orPD-L1; and b. the GITR agonist is an antibody or antigen bindingfragment thereof, that binds GITR.
 3. The method of claim 2 wherein a.the PD-1 antagonist is an antibody or antigen binding fragment thereofthat binds to human PD-1 or PD-L1; and b. the GITR agonist is anantibody or antigen binding fragment thereof that binds to human GITR.4. The method of claim 3, wherein the antibody or binding fragmentthereof is humanized or fully human.
 5. The method of claim 2, whereina. the PD-1 antagonist is selected from the group consisting ofBMS-936558, MK-3475, and MPDL3280A; and b. the GITR agonist is selectedfrom the group consisting of: i. an antibody having at least one CDR ofSEQ ID NOs: 1-66; ii. TRX518; and iii. TRX385.
 6. The method of claim 5,wherein the GITR agonist is an antibody having: a) a heavy chain CDR1 ofSEQ ID NOs: 1-11, CDR2 of SEQ ID NOs: 12-22, and CDR3 of SEQ ID NOs:23-33; and/or b) a light chain CDR1 of SEQ ID NOs: 34-44, CDR2 of SEQ IDNOs: 45-55, and CDR3 of SEQ ID NOs: 56-66.
 7. The method of claim 5,wherein the GITR agonist is an antibody having: a) a variable heavychain of SEQ ID NOs: 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, and 87;and/or b) a variable light chain of SEQ ID NO: 68, 70, 72, 74, 76, 78,80, 82, 84, 86, and
 88. 8. The method of claim 1, wherein the PD-1antagonist and GITR agonist are administered simultaneously at least onetime.
 9. The method of claim 1, wherein the PD-1 antagonist and GITRagonist are administered simultaneously at least 2 times.
 10. The methodof claim 1, wherein the tumor is an advanced stage tumor.
 11. The methodof claim 10, wherein the advanced stage tumor is selected from the groupconsisting of squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer suchas hepatic carcinoma and hepatoma, bladder cancer, breast cancer, coloncancer, colorectal cancer, endometrial carcinoma, myeloma (such asmultiple myeloma), salivary gland carcinoma, kidney cancer such as renalcell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma,prostate cancer, vulval cancer, thyroid cancer, testicular cancer, andesophageal cancer. 12-17. (canceled)
 18. A pharmaceutical combinationcomprising a PD-1 antagonist and a GITR agonist, wherein: a) the PD-1antagonist is selected from the group consisting of BMS-936558, MK-3475,and MPDL3280A; and b) the GITR agonist is selected from the groupconsisting of: i. an antibody having at least one CDR of SEQ ID NOs:1-66; ii. TRX518; and iii. TRX385.
 19. (canceled)
 20. The method ofclaim 5, wherein the GITR agonist is an antibody having: a) a variableheavy chain of SEQ ID NO: 81; and/or b) a variable light chain of SEQ IDNO: 82, wherein amino acid 31 is Q and amino acid 57 is Q.